Many biologically active natural products derive their activity from the sugar components of their structures. Changing the structures of these sugars can have a profound impact on the biological activity, selectivity, and pharmacokinetic properties of the parent compounds. This observation has fueled the development of methods to derivatize natural products with diverse sugar moieties by exploiting the sugar biosynthetic machinery. Fully realizing the potential of such an approach relies on the discovery of new sugar biosynthetic pathways, and also requires a thorough understanding of the biosynthetic pathway of each target sugar including detailed mechanistic knowledge of the key enzymes. With these goals in mind, we have produced notable results in work funded by previous grant. As a result of these studies, we have identified four key areas that warrant further investigation in the next funding period. Accordingly, this application outlines experiments designed to learn how desosamine, apiose, kijanose, and 2-deoxy-2- mercaptoglucose are biosynthesized. The specific objectives include (1) mechanistic studies of a radical-SAM enzyme (DesII) involved in the biosynthesis of desosamine, an essential component of many macrolide antibiotics, (2) a determination of the mechanism of the pyranose-to-furanose ring-contraction catalyzed by UDP-apiose synthase, (3) an investigation into the biosynthesis of the unusual nitrosugar moiety (kijanose) of kijanimicin, and (4) the elucidation of the mechanism of sulfur incorporation into the 2-deoxy-2-mercaptoglucose moiety of the antibiotic BE-7585A. The proposed experiments will not only delineate the biosynthesis of deoxy-, branched-chain, nitro-, and sulfur-containing sugars, but will also advance the field of mechanistic enzymology by enhancing our understanding of several important classes of enzymes. Our results should also be valuable to applied biomedical research, as new glycosylation tools (i.e., sugar biosynthetic pathways and enzymes) will be discovered for future secondary metabolite glycodiversification efforts. PUBLIC HEALTH RELEVANCE: Outlined in this application are experiments designed to study the mechanism of the radical SAM enzyme, DesII, involved in the C4-deoxygenation step in desosamine biosynthesis, the mechanism of the ring contraction reaction catalyzed by UDP-apiose synthase (Axs1), the biosynthesis of an unusual nitrosugar, kijanose, and the mechanism of sulfur incorporation into the thiosugar moiety of BE-7585A. The results are expected to significantly advance the field of mechanistic enzymology and should also be valuable to applied biomedical research, as new sugar biosynthetic pathways and enzymes will be discovered for future secondary metabolite glycodiversification efforts.